Cancerous disease modifying antibodies

ABSTRACT

The present invention relates to a method for producing patient cancerous disease modifying antibodies using a novel paradigm of screening. By segregating the anti-cancer antibodies using cancer cell cytotoxicity as an end point, the process makes possible the production of anti-cancer antibodies for therapeutic and diagnostic purposes. The antibodies can be used in aid of staging and diagnosis of a cancer, and can be used to treat primary tumors and tumor metastases. The anti-cancer antibodies can be conjugated to toxins, enzymes, radioactive compounds, and hematogenous cells.

REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.11/067,390 filed on Feb. 25, 2005, which claims benefit of the filingdate of Provisional Application 60/548,531, filed on Feb. 26, 2004, thecontents of which is herein incorporated by reference.

FIELD OF THE INVENTION

This invention relates to the isolation and production of cancerousdisease modifying antibodies (CDMAB) and to the use of these CDMAB intherapeutic and diagnostic processes, optionally in combination with oneor more chemotherapeutic agents. The invention further relates tobinding assays, which utilize the CDMAB of the instant invention.

BACKGROUND OF THE INVENTION

Each individual who presents with cancer is unique and has a cancer thatis as different from other cancers as that person's identity. Despitethis, current therapy treats all patients with the same type of cancer,at the same stage, in the same way. At least 30 percent of thesepatients will fail the first line therapy, thus leading to furtherrounds of treatment and the increased probability of treatment failure,metastases, and ultimately, death. A superior approach to treatmentwould be the customization of therapy for the particular individual. Theonly current therapy, which lends itself to customization, is surgery.Chemotherapy and radiation treatment cannot be tailored to the patient,and surgery by itself, in most cases is inadequate for producing cures.

With the advent of monoclonal antibodies, the possibility of developingmethods for customized therapy became more realistic since each antibodycan be directed to a single epitope. Furthermore, it is possible toproduce a combination of antibodies that are directed to theconstellation of epitopes that uniquely define a particular individual'stumor.

Having recognized that a significant difference between cancerous andnormal cells is that cancerous cells contain antigens that are specificto transformed cells, the scientific community has long held thatmonoclonal antibodies can be designed to specifically target transformedcells by binding specifically to these cancer antigens; thus giving riseto the belief that monoclonal antibodies can serve as “Magic Bullets” toeliminate cancer cells.

Monoclonal antibodies isolated in accordance with the teachings of theinstantly disclosed invention have been shown to modify the cancerousdisease process in a manner which is beneficial to the patient, forexample by reducing the tumor burden, and will variously be referred toherein as cancerous disease modifying antibodies (CDMAB) or“anti-cancer” antibodies.

At the present time, the cancer patient usually has few options oftreatment. The regimented approach to cancer therapy has producedimprovements in global survival and morbidity rates. However, to theparticular individual, these improved statistics do not necessarilycorrelate with an improvement in their personal situation.

Thus, if a methodology was put forth which enabled the practitioner totreat each tumor independently of other patients in the same cohort,this would permit the unique approach of tailoring therapy to just thatone person. Such a course of therapy would, ideally, increase the rateof cures, and produce better outcomes, thereby satisfying a long-feltneed.

Historically, the use of polyclonal antibodies has been used withlimited success in the treatment of human cancers. Lymphomas andleukemias have been treated with human plasma, but there were fewprolonged remissions or responses. Furthermore, there was a lack ofreproducibility and no additional benefit compared to chemotherapy.Solid tumors such as breast cancers, melanomas and renal cell carcinomashave also been treated with human blood, chimpanzee serum, human plasmaand horse serum with correspondingly unpredictable and ineffectiveresults.

There have been many clinical trials of monoclonal antibodies for solidtumors. In the 1980s there were at least 4 clinical trials for humanbreast cancer which produced only 1 responder from at least 47 patientsusing antibodies against specific antigens or based on tissueselectivity. It was not until 1998 that there was a successful clinicaltrial using a humanized anti-Her2 antibody in combination withCisplatin. In this trial 37 patients were accessed for responses ofwhich about a quarter had a partial response rate and another half hadminor or stable disease progression.

The clinical trials investigating colorectal cancer involve antibodiesagainst both glycoprotein and glycolipid targets. Antibodies such as17-1A, which has some specificity for adenocarcinomas, has undergonePhase 2 clinical trials in over 60 patients with only 1 patient having apartial response. In other trials, use of 17-1A produced only 1 completeresponse and 2 minor responses among 52 patients in protocols usingadditional cyclophosphamide. Other trials involving 17-1A yieldedresults that were similar. The use of a humanized murine monoclonalantibody initially approved for imaging also did not produce tumorregression. To date there has not been an antibody that has beeneffective for colorectal cancer. Likewise there have been equally poorresults for lung cancer, brain cancers, ovarian cancers, pancreaticcancer, prostate cancer, and stomach cancer. There has been some limitedsuccess in the use of anti-GD3 monoclonal antibodies for melanoma. Thus,it can be seen that despite successful small animal studies that are aprerequisite for human clinical trials, the antibodies that have beentested thus far have been, for the most part, ineffective.

Prior Patents:

U.S. Pat. No. 5,750,102 discloses a process wherein cells from apatient's tumor are transfected with MHC genes, which may be cloned fromcells or tissue from the patient. These transfected cells are then usedto vaccinate the patient.

U.S. Pat. No. 4,861,581 discloses a process comprising the steps ofobtaining monoclonal antibodies that are specific to an internalcellular component of neoplastic and normal cells of the mammal but notto external components, labeling the monoclonal antibody, contacting thelabeled antibody with tissue of a mammal that has received therapy tokill neoplastic cells, and determining the effectiveness of therapy bymeasuring the binding of the labeled antibody to the internal cellularcomponent of the degenerating neoplastic cells. In preparing antibodiesdirected to human intracellular antigens, the patentee recognizes thatmalignant cells represent a convenient source of such antigens.

U.S. Pat. No. 5,171,665 provides a novel antibody and method for itsproduction. Specifically, the patent teaches formation of a monoclonalantibody which has the property of binding strongly to a protein antigenassociated with human tumors, e.g. those of the colon and lung, whilebinding to normal cells to a much lesser degree.

U.S. Pat. No. 5,484,596 provides a method of cancer therapy comprisingsurgically removing tumor tissue from a human cancer patient, treatingthe tumor tissue to obtain tumor cells, irradiating the tumor cells tobe viable but non-tumorigenic, and using these cells to prepare avaccine for the patient capable of inhibiting recurrence of the primarytumor while simultaneously inhibiting metastases. The patent teaches thedevelopment of monoclonal antibodies, which are reactive with surfaceantigens of tumor cells. As set forth at col. 4, lines 45 et seq., thepatentees utilize autochthonous tumor cells in the development ofmonoclonal antibodies expressing active specific immunotherapy in humanneoplasia.

U.S. Pat. No. 5,693,763 teaches a glycoprotein antigen characteristic ofhuman carcinomas is not dependent upon the epithelial tissue of origin.

U.S. Pat. No. 5,783,186 is drawn to anti-Her2 antibodies, which induceapoptosis in Her2 expressing cells, hybridoma cell lines producing theantibodies, methods of treating cancer using the antibodies andpharmaceutical compositions including said antibodies.

U.S. Pat. No. 5,849,876 describes new hybridoma cell lines for theproduction of monoclonal antibodies to mucin antigens purified fromtumor and non-tumor tissue sources.

U.S. Pat. No. 5,869,268 is drawn to a method for generating a humanlymphocyte producing an antibody specific to a desired antigen, a methodfor producing a monoclonal antibody, as well as monoclonal antibodiesproduced by the method. The patent is particularly drawn to theproduction of an anti-HD human monoclonal antibody useful for thediagnosis and treatment of cancers.

U.S. Pat. No. 5,869,045 relates to antibodies, antibody fragments,antibody conjugates and single chain immunotoxins reactive with humancarcinoma cells. The mechanism by which these antibodies function istwo-fold, in that the molecules are reactive with cell membrane antigenspresent on the surface of human carcinomas, and further in that theantibodies have the ability to internalize within the carcinoma cells,subsequent to binding, making them especially useful for formingantibody-drug and antibody-toxin conjugates. In their unmodified formthe antibodies also manifest cytotoxic properties at specificconcentrations.

U.S. Pat. No. 5,780,033 discloses the use of autoantibodies for tumortherapy and prophylaxis. However, this antibody is an anti-nuclearautoantibody from an aged mammal. In this case, the autoantibody is saidto be one type of natural antibody found in the immune system. Becausethe autoantibody comes from “an aged mammal”, there is no requirementthat the autoantibody actually comes from the patient being treated. Inaddition the patent discloses natural and monoclonal anti-nuclearautoantibody from an aged mammal, and a hybridoma cell line producing amonoclonal anti-nuclear autoantibody.

SUMMARY OF THE INVENTION

The instant inventors have previously been awarded U.S. Pat. No.6,180,357, entitled “Individualized Patient Specific Anti-CancerAntibodies” directed to a process for selecting individually customizedanti-cancer antibodies, which are useful in treating a cancerousdisease.

This application utilizes, in part, the method for producing patientspecific anti-cancer antibodies as taught in the '357 patent forisolating hybridoma cell lines which encode for cancerous diseasemodifying monoclonal antibodies. These antibodies can be madespecifically for one tumor and thus make possible the customization ofcancer therapy. Within the context of this application, anti-cancerantibodies having either cell killing (cytotoxic) or cell-growthinhibiting (cytostatic) properties will hereafter be referred to ascytotoxic. These antibodies can be used in aid of staging and diagnosisof a cancer, and can be used to treat tumor metastases.

The prospect of individualized anti-cancer treatment will bring about achange in the way a patient is managed. A likely clinical scenario isthat a tumor sample is obtained at the time of presentation, and banked.From this sample, the tumor can be typed from a panel of pre-existingcancerous disease modifying antibodies. The patient will beconventionally staged but the available antibodies can be of use infurther staging the patient. The patient can be treated immediately withthe existing antibodies, and a panel of antibodies specific to the tumorcan be produced either using the methods outlined herein or through theuse of phage display libraries in conjunction with the screening methodsherein disclosed. All the antibodies generated will be added to thelibrary of anti-cancer antibodies since there is a possibility thatother tumors can bear some of the same epitopes as the one that is beingtreated. The antibodies produced according to this method may be usefulto treat cancerous disease in any number of patients who have cancersthat bind to these antibodies.

In addition to anti-cancer antibodies, the patient can elect to receivethe currently recommended therapies as part of a multi-modal regimen oftreatment. The fact that the antibodies isolated via the presentmethodology are relatively non-toxic to non-cancerous cells allows forcombinations of antibodies at high doses to be used, either alone, or inconjunction with conventional therapy. The high therapeutic index willalso permit re-treatment on a short time scale that should decrease thelikelihood of emergence of treatment resistant cells.

Furthermore, it is within the purview of this invention to conjugatestandard chemotherapeutic modalities, e.g. radionuclides, with the CDMABof the instant invention, thereby focusing the use of saidchemotherapeutics.

If the patient is refractory to the initial course of therapy ormetastases develop, the process of generating specific antibodies to thetumor can be repeated for re-treatment. Furthermore, the anti-cancerantibodies can be conjugated to red blood cells obtained from thatpatient and re-infused for treatment of metastases. There have been feweffective treatments for metastatic cancer and metastases usuallyportend a poor outcome resulting in death. However, metastatic cancersare usually well vascularized and the delivery of anti-cancer antibodiesby red blood cells can have the effect of concentrating the antibodiesat the site of the tumor. Even prior to metastases, most cancer cellsare dependent on the host's blood supply for their survival andanti-cancer antibodies conjugated to red blood cells can be effectiveagainst in situ tumors as well. Alternatively, the antibodies may beconjugated to other hematogenous cells, e.g. lymphocytes, macrophages,monocytes, natural killer cells, etc.

There are five classes of antibodies and each is associated with afunction that is conferred by its heavy chain. It is generally thoughtthat cancer cell killing by naked antibodies are mediated either throughantibody-dependent cellular cytotoxicity (ADCC) or complement-dependentcytotoxicity (CDC). For example murine IgM and IgG2a antibodies canactivate human complement by binding the C-1 component of the complementsystem thereby activating the classical pathway of complementactivation, which can lead to tumor lysis. For human antibodies the mosteffective complement-activating antibodies are generally IgM and IgG1.Murine antibodies of the IgG2a and IgG3 isotype are effective atrecruiting cytotoxic cells that have Fc receptors which will lead tocell killing by monocytes, macrophages, granulocytes and certainlymphocytes. Human antibodies of both the IgG1 and IgG3 isotype mediateADCC.

Another possible mechanism of antibody-mediated cancer killing may bethrough the use of antibodies that function to catalyze the hydrolysisof various chemical bonds in the cell membrane and its associatedglycoproteins or glycolipids, so-called catalytic antibodies.

There are two additional mechanisms of antibody-mediated cancer cellkilling which are more widely accepted. The first is the use ofantibodies as a vaccine to induce the body to produce an immune responseagainst the putative cancer antigen that resides on the tumor cell. Thesecond is the use of antibodies to target growth receptors and interferewith their function or to down regulate that receptor so that itsfunction is effectively lost.

The clinical utility of a cancer drug is based on the benefit of thedrug under an acceptable risk profile to the patient. In cancer therapysurvival has generally been the most sought after benefit, however thereare a number of other well-recognized benefits in addition to prolonginglife. These other benefits, where treatment does not adversely affectsurvival, include symptom palliation, protection against adverse events,prolongation in time to recurrence or disease-free survival, andprolongation in time to progression. These criteria are generallyaccepted and regulatory bodies such as the U.S. Food and DrugAdministration (F.D.A.) approve drugs that produce these benefits(Hirschfeld et al. Critical Reviews in Oncology/Hematology 42:137-1432002). In addition to these criteria it is well recognized that thereare other endpoints that may presage these types of benefits. In part,the accelerated approval process granted by the U.S. F.D.A. acknowledgesthat there are surrogates that will likely predict patient benefit. Asof year-end (2003), there have been sixteen drugs approved under thisprocess, and of these, four have gone on to full approval, i.e.,follow-up studies have demonstrated direct patient benefit as predictedby surrogate endpoints. One important endpoint for determining drugeffects in solid tumors is the assessment of tumor burden by measuringresponse to treatment (Therasse et al. Journal of the National CancerInstitute 92(3):205-216 2000). The clinical criteria (RECIST criteria)for such evaluation have been promulgated by Response EvaluationCriteria in Solid Tumors Working Group, a group of international expertsin cancer. Drugs with a demonstrated effect on tumor burden, as shown byobjective responses according to RECIST criteria, in comparison to theappropriate control group tend to, ultimately, produce direct patientbenefit. In the pre-clinical setting tumor burden is generally morestraightforward to assess and document. In that pre-clinical studies canbe translated to the clinical setting, drugs that produce prolongedsurvival in pre-clinical models have the greatest anticipated clinicalutility. Analogous to producing positive responses to clinicaltreatment, drugs that reduce tumor burden in the pre-clinical settingmay also have significant direct impact on the disease. Althoughprolongation of survival is the most sought after clinical outcome fromcancer drug treatment, there are other benefits that have clinicalutility and it is clear that tumor burden reduction can also lead todirect benefits and have clinical impact (Eckhardt et al. DevelopmentalTherapeutics: Successes and Failures of Clinical Trial Designs ofTargeted Compounds; ASCO Educational Book, 39^(th) Annual Meeting, 2003,pages 209-219).

Accordingly, it is an objective of the invention to utilize a method forproducing CDMAB from cells derived from a particular individual whichare cytotoxic with respect to cancer cells while simultaneously beingrelatively non-toxic to non-cancerous cells, in order to isolatehybridoma cell lines and the corresponding isolated monoclonalantibodies and antigen binding fragments thereof for which saidhybridoma cell lines are encoded.

It is an additional objective of the invention to teach CDMAB andantigen binding fragments thereof.

It is a further objective of the instant invention to produce CDMABwhose cytotoxicity is mediated through antibody dependent cellulartoxicity.

It is yet an additional objective of the instant invention to produceCDMAB whose cytotoxicity is mediated through complement dependentcellular toxicity.

It is still a further objective of the instant invention to produceCDMAB whose cytotoxicity is a function of their ability to catalyzehydrolysis of cellular chemical bonds.

A still further objective of the instant invention is to produce CDMAB,which are useful in a binding assay for diagnosis, prognosis, andmonitoring of cancer.

Other objects and advantages of this invention will become apparent fromthe following description wherein, by way of illustration and example,certain embodiments of this invention are set forth.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 includes representative FACS histograms of AR40A603.13 andanti-EGFR antibodies directed against several cancer and non-cancer celllines;

FIG. 2 compares the percentage cytotoxicity of the hybridomasupernatants against cell lines PC-3, LnCap and CCD-27sk versus bindinglevels;

FIG. 3 is a comparison of cytotoxicity of AR40A603.13 versus positiveand negative controls;

FIG. 4 represents binding of AR40A603.13 versus anti-EGFR control andtabulated the mean fluorescence intensity fold increase above isotypecontrol. Results are presented qualitatively as: between 1.5 to 5 (+); 5to 25 (++); 25 to 50 (+++); and above 50 (++++);

FIG. 5 demonstrates the effect of AR40A603.13 on tumor growth in aSW1116 colon cancer model. The vertical lines indicate the period duringwhich the antibody was administered. Data points represent themean+/−SEM;

FIG. 6 demonstrates the effect of AR40A603.13 on body weight in a SW1116colon cancer model. Data points represent the mean+/−SEM;

FIG. 7 demonstrates the effect of AR40A603.13 on tumor growth in a PC-3colon cancer model. The vertical lines indicate the period during whichthe antibody was administered. Data points represent the mean+/−SEM;

FIG. 8. demonstrates the effect of AR40A603.13 on body weight in a PC-3prostate cancer model. Data points represent the mean+/−SEM.

DETAILED DESCRIPTION OF THE INVENTION Example 1 Hybridoma ProductionHybridoma Cell Line AR40A603.13

The hybridoma cell line AR40A603.13 was deposited, in accordance withthe Budapest Treaty, with the International Depository Authority ofCanada (IDAC), Bureau of Microbiology, Health Canada, 1015 ArlingtonStreet, Winnipeg, Manitoba, Canada, R3E 3R2, on Jan. 28, 2004, underAccession Number 280104-04. In accordance with 37 CFR 1.808, thedepositors assure that all restrictions imposed on the availability tothe public of the deposited materials will be irrevocably removed uponthe granting of a patent.

To produce the hybridoma that produces the anti-cancer antibodyAR40A603.13, a single cell suspension of frozen human prostate tumortissue (Genomics Collaborative, Cambridge, Mass.) was prepared in PBS.IMMUNEASY™ (Qiagen, Venlo, Netherlands) adjuvant was prepared for use bygentle mixing. Five to seven week old BALB/c mice were immunized byinjecting subcutaneously, 2 million cells in 50 microliters of theantigen-adjuvant. Recently prepared antigen-adjuvant was used to boostthe immunized mice intraperitoneally, 2 and 5 weeks after the initialimmunization, with 2 million cells in 50 microliters. A spleen was usedfor fusion three days after the last immunization. The hybridomas wereprepared by fusing the isolated splenocytes with NSO-1 myeloma partners.The supernatants from the fusions were tested for subcloning of thehybridomas.

To determine whether the antibodies secreted by the hybridoma cells areof the IgG or IgM isotype, an ELISA assay was employed. 100microliters/well of goat anti-mouse IgG+IgM (H+L) at a concentration of2.4 micrograms/mL in coating buffer (0.1M carbonate/bicarbonate buffer,pH 9.2-9.6) at 4° C. was added to the ELISA plates overnight. The plateswere washed thrice in washing buffer (PBS+0.05% Tween). 100microliters/well blocking buffer (5% milk in wash buffer) was added tothe plate for 1 hr. at room temperature and then washed thrice inwashing buffer. 100 microliters/well of hybridoma supernatant was addedand the plate incubated for 1 hr. at room temperature. The plates werewashed thrice with washing buffer and 1/100,000 dilution of either goatanti-mouse IgG or IgM horseradish peroxidase conjugate (diluted in PBScontaining 5% milk), 100 microliters/well, was added. After incubatingthe plate for 1 hr. at room temperature the plate was washed thrice withwashing buffer. 100 microliters/well of TMB solution was incubated for1-3 minutes at room temperature. The color reaction was terminated byadding 100 microliters/well 2M H₂SO₄ and the plate was read at 450 nmwith a Perkin-Elmer HTS7000 plate reader. As indicated in FIG. 2, theAR40A603.13 hybridoma secreted primarily antibodies of the IgG isotype.

After one round of limiting dilution hybridoma supernatants were testedfor antibodies that bound to target cells in a cell ELISA assay. Twohuman prostate cancer cell lines and 1 human normal skin cell line weretested: PC-3, LnCap and CCD-27sk respectively. The plated cells werefixed prior to use. The plates were washed thrice with PBS containingMgCl₂ and CaCl₂ at room temperature. 100 microliters of 2%paraformaldehyde diluted in PBS was added to each well for 10 minutes atroom temperature and then discarded. The plates were again washed withPBS containing MgCl₂ and CaCl₂ three times at room temperature. Blockingwas done with 100 microliters/well of 5% milk in wash buffer (PBS+0.05%Tween) for 1 hr at room temperature. The plates were washed thrice withwash buffer and the hybridoma supernatant was added at 100microliters/well for 1 hr at room temperature. The plates were washed 3times with wash buffer and 100 microliters/well of 1/25,000 dilution ofgoat anti-mouse IgG or IgM antibody conjugated to horseradish peroxidase(diluted in PBS containing 5% milk) was added. After 1 hr incubation atroom temperature the plates were washed 3 times with wash buffer and 100microliter/well of TMB substrate was incubated for 1-3 minutes at roomtemperature. The reaction was terminated with 100 microliters/well 2MH₂SO₄ and the plate read at 450 nm with a Perkin-Elmer HTS7000 platereader. The results as tabulated in FIG. 2 were expressed as the numberof folds above background compared to an in-house IgG isotype controlthat has previously been shown not to bind to the cell lines tested. Theantibodies from the hybridoma AR40A603.13 showed substantial binding tothe prostate cancer cell line LnCap, followed by another prostate cancercell line PC-3. AR40A603.13 displayed the lowest level of binding to thenormal skin cell line CCD-27sk.

In conjunction with testing for antibody binding the cytotoxic effect ofthe hybridoma supernatants were tested in the same cell lines: PC-3,LnCap and CCD-27sk. The Live/Dead cytotoxicity assay was obtained fromMolecular Probes (Eu, OR). The assays were performed according to themanufacturer's instructions with the changes outlined below. Cells wereplated before the assay at the predetermined appropriate density. After2 days, 100 μl of supernatant from the hybridoma microtitre plates weretransferred to the cell plates and incubated in a 5 percent CO₂incubator for 5 days. The wells that served as the positive controlswere aspirated until empty and 100 μl of sodium azide (NaN₃) orcycloheximide was added. After 5 days of treatment, the plates were thenemptied by inverting and blotting dry. Room temperature DPBS (Dulbecco'sphosphate buffered saline) containing MgCl₂ and CaCl₂ was dispensed intoeach well from a multichannel squeeze bottle, tapped 3 times, emptied byinversion and then blotted dry. 50 μl of the fluorescent calcein dyediluted in DPBS containing MgCl₂ and CaCl₂ was added to each well andincubated at 37° C. in a 5% CO₂ incubator for 30 minutes. The plateswere read in a Perkin-Elmer HTS7000 fluorescence plate reader and thedata was analyzed in Microsoft Excel. The results are tabulated in FIG.2. The AR40A603.13 hybridoma produced specific cytotoxicity of 13percent on both the PC-3 and LnCap cells, which was 24 and 22 percent ofthe cytotoxicity obtained in the PC-3 cells and likewise, 16 and 42percent in the LnCap cells, with the positive controls sodium azide andcycloheximide respectively. Results from FIG. 2 demonstrated that thecytotoxic effects of AR40A603.13 were not proportional to the bindinglevels on the two cancer cell types. There was the same level ofcytotoxicity produced in both the LnCap and PC-3 cells. However, therewas at least a three-fold increase in binding of AR40A603.13 to LnCapversus PC-3 cells. As tabulated in FIG. 2, AR40A603.13 did not producecytotoxicity in the CCD-27sk normal cell line despite substantialbinding to these cells. The known non-specific cytotoxic agentscycloheximide and NaN₃ generally produced cytotoxicity as expected.

Example 2 Antibody Production

AR40A603.13 monoclonal antibody was produced by culturing the hybridomain CL-1000 flasks (BD Biosciences, Oakville, ON) with collections andreseeding occurring twice/week. Standard antibody purificationprocedures with Protein G Sepharose 4 Fast Flow (Amersham Biosciences,Baie d'Urfé, QC) were followed. It is within the scope of this inventionto utilize monoclonal antibodies that are humanized, chimerized ormurine.

AR40A603.13 was compared to a number of both positive (anti-EGFR (C225,IgG1, kappa, 5 μg/mL, Cedarlane, Hornby, ON), cycloheximide (CHX, 0.5μM, Sigma, Oakville, ON), and NaN₃ (0.1%, Sigma, Oakville, ON)) andnegative (107.3 (anti-TNP), IgG1, kappa, 20 μg/mL, BD Bioscience,Oakville, ON), and IgG Buffer (3%)) controls in a cytotoxicity assay(FIG. 3). Breast (MDA-MB-231 (MB-231), NCI-MCF-7 (MCF-7)), colon (DLD-1,Lovo, SW1116), ovarian (OVCAR-3), pancreatic (BxPC-3), and prostate(PC-3, LnCap, DU-145) cancer, and non-cancer skin (CCD-27sk), and lung(Hs888.Lu) cell lines were tested (all from the ATCC, Manassas, Va.).The Live/Dead cytotoxicity assay was obtained from Molecular Probes(Eugene, Oreg.). The assays were performed according to themanufacturer's instructions with the changes outlined below. Cells wereplated before the assay at the predetermined appropriate density. After2 days, 100 μl of purified antibody or controls were diluted into media,and then transferred to the cell plates and incubated in a 5 percent CO₂incubator for 5 days. The plates were then emptied by inverting andblotted dry. Room temperature DPBS containing MgCl₂ and CaCl₂ wasdispensed into each well from a multichannel squeeze bottle, tapped 3times, emptied by inversion and then blotted dry. 50 μl of thefluorescent calcein dye diluted in DPBS containing MgCl₂ and CaCl₂ wasadded to each well and incubated at 37° C. in a 5 percent CO₂ incubatorfor 30 minutes. The plates were read in a Perkin-Elmer HTS7000fluorescence plate reader and the data was analyzed in Microsoft Exceland the results were tabulated in FIG. 3. The data was represented as anaverage of four experiments tested in triplicate and is presentedqualitatively in the following fashion: 3/4 to 4/4 experiments with >15%cytotoxicity above background (++++), 2/4 experiments with >15%cytotoxicity above background (+++), at least 2/4 experiments with10-15% cytotoxicity above background (++), at least 2/4 experiments with8-10% cytotoxicity above background (+), 7% cytotoxicity abovebackground (+/−). Unmarked cells in FIG. 3 represented inconsistent oreffects less than the threshold cytotoxicity. The AR40A603.13 antibodyproduced cytotoxicity in the SW1116 colon cancer cell line, the MCF-7breast cancer cell line, the OVCAR-3 ovarian cancer cell line, the LnCapprostate cancer cell line and the BxPC-3 pancreatic cancer cell linerelative to both isotype and buffer negative controls. Importantly,AR40A603.13 did not produce significant cytotoxicity, above negativecontrols, against non-cancer cell lines such as CCD-27sk or Hs888.Lu,indicating that the antibody has specificity towards cancer cells. Thechemical cytotoxic agents induced their expected non-specificcytotoxicity.

Binding of AR40A603.13 to the above-mentioned panel of cancer and normalcell lines was assessed by flow cytometry (FACS). Cells were preparedfor FACS by initially washing the cell monolayer with DPBS (without Ca⁺⁺and Mg⁺⁺). Cell dissociation buffer (INVITROGEN, Burlington, ON) wasthen used to dislodge the cells from their cell culture plates at 37° C.After centrifugation and collection, the cells were resuspended in DPBScontaining MgCl₂, CaCl₂ and 2 percent fetal bovine serum at 4° C.(staining media) and counted, aliquoted to appropriate cell density,spun down to pellet the cells and resuspended in staining media at 4° C.in the presence of test antibodies (AR40A603.13) or control antibodies(isotype control, anti-EGFR) at 20 μg/mL on ice for 30 minutes. Prior tothe addition of Alexa Fluor 488-conjugated secondary antibody the cellswere washed once with staining media. The Alexa Fluor 488-conjugatedantibody in staining media was then added for 30 minutes. The cells werethen washed for the final time and resuspended in fixing media (stainingmedia containing 1.5% paraformaldehyde). Flow cytometric acquisition ofthe cells was assessed by running samples on a FACScan using theCellQuest software (BD Biosciences, Oakville, ON). The forward (FSC) andside scatter (SSC) of the cells were set by adjusting the voltage andamplitude gains on the FSC and SSC detectors. The detectors for thefluorescence (FITC) channel was adjusted by running cells stained onlywith Alexa Fluor 488-conjugated secondary antibody such that cells had auniform peak with a median fluorescent intensity of approximately 1-5units. For each sample, approximately 10,000 stained fixed cells wereacquired for analysis and the results are presented in FIG. 4.

FIG. 4 tabulated the mean fluorescence intensity fold increase aboveisotype control and is presented qualitatively as: between 1.5 to 5 (+);5 to 25 (++); 25 to 50 (+++); and above 50 (++++). Representativehistograms of AR40A603.13 antibodies were compiled for FIG. 1.AR40A603.13 showed binding to all cell lines tested. These data havedemonstrated that AR40A603.13 exhibited functional specificity in thatalthough there was clear binding to all cancer types tested, there wasonly associated cytotoxicity with SW1116, MCF-7, OVCAR-3, LnCap andBxPC-3 cancer cells types. By contrast, the anti-EGFR antibody displayeda higher correlation between binding and cytotoxicity with one suchexample being the non-cancer epidermis derived cell line, CCD-27sk.AR40A603.13 was also effective in several cancer cell lines that werenot affected by anti-EGFR, including SW1116, BxPC-3 and MCF-7.

Example 3 In Vivo SW116 Tumor Experiments

With reference to FIGS. 5 and 6, 4 to 8 week old female SCID mice wereimplanted with 5 million human colon cancer cells (SW116) in 100microlitres saline injected subcutaneously in the scruff of the neck.The mice were randomly divided into 2 treatment groups of 5. On the dayafter implantation, 20 mg/kg of AR40A603.13 test antibody or buffercontrol was administered intraperitoneally at a volume of 300microliters after dilution from the stock concentration with a diluentthat contained 2.7 mM KCl, 1 mM KH₂PO₄, 137 mM NaCl and 20 mM Na₂HPO₄.The antibodies were then administered once per week for a period of 7weeks in the same fashion. Tumor growth was measured about every seventhday with calipers for up to 8 weeks or until individual animals reachedthe Canadian Council for Animal Care (CCAC) end-points. Body weights ofthe animals were recorded for the duration of the study. At the end ofthe study all animals were euthanised according to CCAC guidelines.

AR40A603.13 prevented tumor growth and reduced tumor burden in apreventative in vivo model of human colon cancer. On day 55post-implantation, 5 days after the last treatment dose, the mean tumorvolume in the AR40A603.13 treated group was 71 percent of the tumorvolume in the buffer control-treated group (p=0.0493, t-test, FIG. 5).

There were no clinical signs of toxicity throughout the study. Bodyweight measured at weekly intervals was a surrogate for well-being andfailure to thrive. As seen in FIG. 6, there was no significantdifference in body weight between the groups at the end of the treatmentperiod (p=0.1152, t-test). Within groups, the body weight of the buffertreated control animals did not vary significantly between the start andend of the study period (p=0.2825, t-test), while the body weight of theAR40A603.13 treated group showed a slight significant increase from amean of 17.80 g to 19.80 g (p=0.0345, t-test). Therefore AR40A603.13 waswell-tolerated and decreased the tumor burden in a colon cancerxenograft model.

Example 4 In Vivo PC-3 Tumor Experiments

With reference to FIGS. 7 and 8, 4 to 8 week old male SCID mice wereimplanted with 1 million human prostate cancer cells (PC-3) in 100microlitres saline injected subcutaneously in the scruff of the neck.The mice were randomly divided into 2 treatment groups of 5. On the dayafter implantation, 20 mg/kg of AR40A603.13 test antibody or buffercontrol was administered intraperitoneally at a volume of 300microliters after dilution from the stock concentration with a diluentthat contained 2.7 mM KCl, 1 mM KH₂PO₄, 137 mM NaCl and 20 mM Na₂HPO₄.The antibodies were then administered once per week for the duration ofthe study in the same fashion. Tumor growth was measured about everyseventh day with calipers. The study was completed after 6 injections(41 days), as the animals reached the Canadian Council for Animal Care(CCAC) end-points due to large ulcerated lesions. Body weights of theanimals were recorded for the duration of the study. At the end of thestudy all animals were euthanised according to CCAC guidelines.

AR40A603.13 prevented tumor growth and reduced tumor burden in apreventative in vivo model of human prostate cancer. On day 41post-implantation, 5 days after the last treatment dose, the mean tumorvolume in the AR40A603.13 treated group was 60 percent of the tumorvolume in the buffer control-treated group (p=0.055, t-test, FIG. 7).

In a PC-3 prostate cancer xenograft model, body weight can be used as asurrogate indicator of disease progression (Wang et al. Int J Cancer,2003). As seen in FIG. 8, by the end of the study, day 41, controlanimals exhibited a 27% decrease in body weight from the onset of thestudy. By contrast, the group treated with AR40A603.13 had asignificantly higher body weight than the control group (p=0.0465,t-test). Overall, the AR40A603.13-treated group lost 36% less weightthan the buffer control group. Therefore AR40A603.13 was well-toleratedand decreased the tumor burden and cachexia in a prostate cancerxenograft model.

REFERENCE

-   Wang Z, Corey E, Hass G M, et al. Expression of the human    cachexia-associated protein (HCAP) in prostate cancer and in a    prostate cancer animal model of cachexia. Int J Cancer. 2003;    105(1): 123-9.

1. An isolated monoclonal antibody encoded by the clone deposited withthe IDAC as Accession Number 280104-04.
 2. The antibody of claim 1,which is humanized.
 3. The antibody or of claim 1, which is chimerized.4. The isolated clone deposited with the IDAC as Accession Number280104-04.
 5. A method for initiating antibody induced cellularcytotoxicity of cancerous cells in a tissue sample selected from a humantumor comprising: providing a tissue sample from said human tumor;providing an isolated monoclonal antibody encoded by the clone depositedwith the IDAC as Accession Number 280104-04 or a cellular cytotoxicityinducing antigen binding fragment thereof; and contacting said isolatedmonoclonal antibody or cellular cytotoxicity inducing antigen bindingfragment thereof with said tissue sample.
 6. The method of claim 5wherein the human tumor tissue sample is obtained from a tumororiginating in a tissue selected from the group consisting of colon,ovarian, prostate, pancreatic and breast tissue.
 7. Antigen bindingfragments of the isolated monoclonal antibody of claim
 1. 8. Antigenbinding fragments of the humanized antibody of claim
 2. 9. Antigenbinding fragments of the chimerized antibody of claim
 3. 10. Theisolated antibody or antigen binding fragments of any one of claims 1,2, 3, 7, 8 or 9 conjugated with a member selected from the groupconsisting of cytotoxic moieties, enzymes, radioactive compounds, andhematogenous cells.
 11. A method of treating a human tumor susceptibleto antibody induced cellular cytotoxicity in a mammal, wherein saidhuman tumor expresses an antigen which specifically binds to themonoclonal antibody which has the cellular cytotoxicity inducingcharacteristics of the monoclonal antibody encoded by a clone depositedwith the IDAC as Accession Number 280104-04 or a cellular cytotoxicityinducing antigen binding fragment thereof, comprising administering tosaid mammal said monoclonal antibody or said antigen binding fragmentthereof in an amount effective to induce cellular cytotoxicity andthereby reduce said mammal's tumor burden.
 12. The method of claim 11wherein said monoclonal antibody is conjugated to a cytotoxic moiety.13. The method of claim 12 wherein said cytotoxic moiety is aradioactive isotope.
 14. The method of claim 11 wherein said monoclonalantibody activates complement.
 15. The method of claim 11 wherein saidmonoclonal antibody mediates antibody dependent cellular cytotoxicity.16. The method of claim 11 wherein said monoclonal antibody ishumanized.
 17. The method of claim 11 wherein said monoclonal antibodyis chimerized.